J. Embryol. exp. Morph. 92, 133-143 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
133
Proximal—distal sequence of development of the
skeletal tissues in the penis of rat and the inductive
effect of epithelium
RYUTARO MURAKAMI AND TAKEO MIZUNO
Zoological Institute, Faculty of Science, University of Tokyo, Tokyo 113, Japan
SUMMARY
The penis of adult rats comprises the corpus cavernosum penis and the proximal and distal
segments of os penis which are situated proximal-distally in this order. Androgens are necessary
for the phenotypic differentiation of these tissues. In the present study, genital tubercular
mesenchyme of foetal rats was recombined with or without homologous or heterologous
epithelia and transplanted beneath the kidney capsule of syngeneic adult male rats, and the
development of the corpus cavernosum penis and the os penis in the transplants was examined.
Only in the presence of epithelia can the genital tubercular mesenchyme acquire the capacity for
differentiation into the corpus cavernosum penis, the proximal segment of os penis, and the
distal segment of os penis in this chronological order. The inductive effect of epithelium is a
permissive step in the differentiation of the os penis of rat. The epithelium seems to be necessary
for the process of rudiment formation of the os penis and the corpus cavernosum penis.
INTRODUCTION
The penis of the rat possesses a skeletal system chiefly comprising the corpus
cavernosum penis and the os penis. Mesenchymal cells in the genital tubercle
of rat foetuses form the rudiment of the corpus cavernosum penis, the rudiment of
the proximal segment (p-segment) of os penis, and the rudiment of the distal
segment (d-segment) of os penis in proximal-distal arrangement. In the male rat,
the rudiment of the corpus cavernosum penis develops into an erectile tissue
possessing lacunae and trabeculae, the rudiment of the p-segment into a membrane bone in its distal half and a hyaline cartilage in its proximal half, and the
rudiment of the d-segment into a fibrocartilage responding to androgens after birth
(Fig. 1) (Glucksmann & Cherry, 1972; Beresford & Burkart, 1977; Beresford &
Clayton, 1977; Yoshida, Kadota & Fukunishi, 1980; Murakami & Mizuno,
1984a,b). The chondrogenesis and osteogenesis in the os penis and the erectile
tissue formation in the corpus cavernosum penis of rats are dependent on
androgens, while the rudiments of these tissues can be formed without androgens
(Murakami, in preparation).
* Present address: Biological Institute, Faculty of Science, Yamaguchi University, Yamaguchi
753, Japan.
Key words: os penis, epithelial-mesenchymal interaction, chondrogenesis, osteogenesis,
androgens, genital tubercle.
134
R. MURAKAMI AND T. MIZUNO
Mesenchymal cells in various organs in vertebrate embryos need inductive
interaction with the epithelium to differentiate into cartilages and bones when
cultured in situ, in vivo, or in vitro. The inductive effects of epithelium have been
reported in the development of the cartilages in limb bud (Saunders, 1948;
Gumpel-Pinot, 1972), scleral cartilage (Newsome, 1972), and membrane bone in
mandible (Tyler & Hall, 1977). Especially in the limb bud, the progenitor cells of
cartilages need the apical ectodermal ridge (AER) to acquire the potency of
differentiation, and the chondrogenic potency is acquired first by the progenitor
cells of the most proximal part of the future limb, then by the progenitor cells of
the more distal parts, that is, in proximal-distal sequence (Saunders, 1948;
Summerbell, 1974). In order to understand the processes of skeletal development
and the role of epithelial-mesenchymal interactions in this process, it is necessary
to determine whether or not development of the skeletal tissues in the penis of rat
proceeds in proximal-distal sequence, and whether or not the epithelialmesenchymal interaction is necessary for these tissues to acquire the potency of
differentiation. Male and female genital tubercles transplanted beneath the kidney
capsule of adult male rats can form the corpus cavernosum penis, the p- and dsegments of os penis preserving their normal arrangement (Murakami, in
preparation). In the present study, the genital tubercular mesenchyme of foetal
rats was recombined with or without epithelia and transplanted beneath the
kidney capsule of adult male rats, and development of the corpus cavernosum
penis and the os penis in the transplants was examined.
MATERIALS AND METHODS
Inbred Wistar Lewis rats and inbred Wistar/Tw rats were used for the transplantation
experiments, and Wistar Imamichi rats were also used for study of the normal histology of the
U
Fig. 1. Schematic illustration of the longitudinal section of the skeletal tissues in the
penis of the 4-week-old male rat. The distal end of the penis is on the right, and the
proximal part is on the left of the picture, c, corpus cavernosum penis; eg, corpus
cavernosum glandis; d, distal segment of os penis; h, hyaline cartilage; m, membrane
bone; p, proximal segment of os penis; u, urethra.
Induction of os penis
135
penis, which was similar to that of Wistar Lewis and Wistar/ Tw strains. The animals were mated
during the night and copulation was confirmed by the presence of spermatozoa in the vaginal
smear next morning. The conceptus was designated as 0-5 days old at 12:00 of this day. Genital
tubercles were excised from male and female foetuses at 14-5-18-5 days of gestation, and treated
with 0-02% collagenase (Worthington Biochemicals Co., Code CLS) dissolved in saline for
30-50 min at room temperature. Urethral epithelium and surface epithelium of genital tubercles
were removed with forceps, and the isolated mesenchyme was washed with 50% foetal bovine
serum in saline for 30-50 min at room temperature. For control explants, the surface epithelium
was not removed, or was removed and recombined with isolated mesenchyme of the genital
tubercle. These two kinds of control explants brought about the same results. The recombined
explants were attached to a Millipore filter (pore size 1-2 jum), placed on a stainless steel grid in a
small glass dish, incubated in BGJb medium (Biggers, Gwatkin & Heyner, 1961) supplemented
with 20% foetal bovine serum and 100 jug ascorbic acid ml"1, and cultured at 37 °C for 1 day to
ensure adhesion between mesenchyme and epithelium. Explants with or without epithelium
were transplanted beneath kidney capsule of syngeneic adult male rats (ages 3-8 months) under
anaesthesia of the hosts with 25mg Nembutal (Abbot Laboratories, 111., USA) per kg b.w. For
examination of the specificity of the epithelial effect, dorsal epidermis and urinary bladder
epithelium of 16-5-day foetuses and urinary bladder epithelium of adult rats were isolated by the
collagenase treatment and washed with 50% foetal bovine serum in saline and recombined
with the isolated genital tubercular mesenchyme of 16-5-day foetuses. The heterologous
recombination with the mesenchymes at earlier stages could not be performed because of the
technical difficulties. The recombinates were cultured in vitro for 1 day, and transplanted in adult
males. 2 or 3 weeks after transplantation, the transplants were fixed with neutral formalin,
decalcified with formic acid (Morse, 1945), and embedded in paraffin. The sections of the
transplants were stained with Alcian blue (pHl-O)-haematoxylin-eosin. Normal histology of
the skeletal tissues in the penis was also studied by the same procedure.
RESULTS
Normal histology of the skeletal tissues in the penis
Normal development of the os penis of the rat was described in detail in the
previous paper (Murakami & Mizuno, 1984a). Briefly, the penis of the rat has the
corpus cavernosum penis, the p-segment of the os penis, and the d-segment of the
os penis, which are situated proximal-distally in this order (Fig. 1). The rudiments
of these tissues are formed as dense mesenchymal cell masses in the genital
tubercles of male and female foetuses at 16-5-18-5 days of gestation (Fig. 2). The
corpus cavernosum penis is an erectile tissue possessing lacunae and trabeculae
(Fig. 3) and begins to be formed at about 1 week after birth. The p-segment of the
os penis is a Haversian bone with a hyaline cartilage at its proximal end (Fig. 4).
The p-segment is formed by fusion of a membrane bone and a hyaline cartilage
within 1 week after birth, and grows successively by endochondral ossification.
The d-segment consists of a fibrocartilage (Fig. 5) which is formed at about 4
weeks after birth and ossified gradually from about 10 weeks after birth.
Development of genital tubercular mesenchyme cultivated in recombination with
homologous epithelium
We used genital tubercles at five developmental stages: 14-5, 15-5, 16-5, 17-5
and 18-5 days of gestation. At 14-5 and 15-5 days of gestation, neither the rudiment
of the corpus cavernosum penis nor that of the os penis was yet formed. At 16-5
days, mesenchymal condensation began in the ventral side of the urethra. At 17-5
136
R. M U R A K A M I AND T.
MIZUNO
Figs 2-5. Normal histology of the skeletal tissues in the penis of the rat.
Fig. 2. Longitudinal section of the genital tubercle of an 18-5-day male foetus. The
proximal end is on the left, and the distal end is on the right. The rudiments of the
corpus cavernosum penis (c), the proximal (p), and the distal (rf) segments of os penis
were recognized as dense mesenchymal cell masses. x64.
Fig. 3. Section of the corpus cavernosum penis of a 2-week-old male. xl70.
Fig. 4. Longitudinal section of the p-segment of os penis of an 1-week-old male. The
proximal end is on the left. The hyaline cartilage (h) and the membrane bone (m) fused
together. Note the beginning of the endochondral ossification. x94.
Fig. 5. Section of the fibrocartilage of the d-segment of os penis of an 8-week-old
male. X170.
Fig. 6. Longitudinal section of the genital tubercular mesenchyme of a 17-5-day foetus
recombined with the homologous epithelium, transplanted in an adult male rat, and
cultivated for 3 weeks. The corpus cavernosum penis (c), the hyaline cartilage (h) and
the membrane bone (m) of the p-segment, and the fibrocartilage of the d-segment (d)
of os penis differentiated preserving the arrangement seen in normal males in situ.
xllO.
Fig. 7. Section of the genital tubercular mesenchyme of a 14-5-day foetus transplanted in an adult male rat and cultivated for 3 weeks. A small erectile tissue is
assumed to be a part of corpus cavernosum penis. xl70.
Fig. 8. Section of the genital tubercular mesenchyme of a 15-5-day foetus transplanted in an adult male rat and cultivated for 3 weeks. Only a large erectile tissue of
corpus cavernosum penis differentiated. x230.
Induction ofos penis
137
138
R. MURAKAMI AND T. MIZUNO
and 18-5 days, the rudiment of the corpus cavernosum penis and the rudiments of
the p- and d-segments of os penis were recognized as dense mesenchymal cell
masses (Fig. 2). Since the developmental fate of transplants of both sexes was
similar, the following results are described without distinguishing the sex of
donors.
The transplants of mesenchyme recombined with epithelium became much
larger than those of mesenchyme alone during in vivo cultivation. Within 2 or 3
weeks after transplantation, mesenchymes of recombinates taken from all the
stages examined formed corpus cavernosum penis, a hyaline cartilage and a
membrane bone of the p-segment of os penis, and a fibrocartilage of the d-segment
of os penis in the same arrangement as that in normal males (Fig. 6, compare with
Figs 1, 2-5). In some transplants, hyaline cartilage and membrane bone of the
p-segment fused immediately after differentiation, as seen in normal males, while
in other transplants, neither component of the p-segment fused, at least during the
course of the experiments.
Development of genital tubercular mesenchyme cultured in the absence of the
epithelium
The transplanted genital tubercular mesenchyme of 14-5-day foetuses formed
only a small erectile tissue assumed to be a part of the corpus cavernosum penis
and/or the connective tissue possessing no specialized structure (Fig. 7). The
mesenchyme of 15-5-day foetuses formed a well-developed corpus cavernosum
penis, while neither the os penis nor its rudiments were formed in transplants
(Fig. 8). Mesenchyme of 16-5-day foetuses developed into corpus cavernosum
penis and hyaline cartilage of the p-segment of os penis (Fig. 9), and, with a
low frequency, membrane bone of the p-segment (Fig. 10), while neither the
d-segment of os penis nor its rudiment were formed (Figs 9, 10). These transplants had a histological appearance as if they were truncated at the level of the
p-segment of os penis (Figs 9, 10). The developmental fate of transplanted
mesenchyme of 17-5- and 18-5-day foetuses was similar to that of mesenchyme
recombined with epithelium, that is, corpus cavernosum penis, hyaline cartilage
and membrane bone of the p-segment, and fibrocartilage of the d-segment of os
penis were formed in the same arrangement as in normal males (Fig. 11). The
Fig. 9. Longitudinal section of the genital tubercular mesenchyme of a 16-5-day foetus
transplanted in an adult male rat and cultivated for 3 weeks. Corpus cavernosum penis
(c) and the hyaline cartilage (h) of the p-segment of os penis differentiated, x 170.
Fig. 10. Longitudinal section of another genital tubercular mesenchyme of a 16-5-day
foetus transplanted in an adult male rat and cultivated for 3 weeks. Corpus cavernosum
penis (c), the hyaline cartilage (h) and the membrane bone (m) of the p-segment
differentiated. xllO.
Fig. 11. Longitudinal section of the genital tubercular mesenchyme of a 17-5-day foetus
transplanted in an adult male rat and cultivated for 3 weeks. Corpus cavernosum penis
(c), the hyaline cartilage (h) and membrane bone (m) of the p-segment, and the
fibrocartilage of the d-segment (d) of os penis differentiated preserving the
arrangement seen in normal males in situ. xllO.
Induction ofos penis
139
140
R. MURAKAMI AND T. MIZUNO
Table 1. Development of corpus cavernosum penis (c) and os penis in the genital
tubercular mesenchyme recombined with or without homologous epithelium
Transplants with differentiated tissues (%)
Membrane
bone
D-segment of
os penis
Fibrocartilage
79
44
76
82
86
100
100
100
0
0
0
0
P-segment of os penis
Foetal age
(days)
No. of
transplants
c
14-5
15-5
16-5
17-5
14
18
27
17
92
100
100
100
14-5
15-5
16-5
17-5
18-5
14
20
55
100
0
0
31
100
55
10
0
15
5
100
100
93
100
73
100
100
100
Hyaline
cartilage
With epithelium
43
39
44
87
Without epithelium
c, corpus cavernosum penis.
Table 2. Development of corpus cavernosum penis (c) and os penis in the genital
tubercular mesenchyme of 16-5-day foetus recombined with heterologous epithelium
Transplants with differentiated tissues (%)
P-segment of os penis
Origin of
epithelium
D-segment of
No. of
transplants
c
Hyaline
cartilage
Membrane
bone
"
Fibrocartilage
31
27
100
100
55
44
10
76
0
100
11
100
73
18
100
9
100
56
22
78
5
100
60
0
100
None
Genital tubercle of
16-5-day foetus
Dorsal skin of 16-5-day
foetus
Urinary bladder of
16-5-day foetus
Urinary bladder of adult
c, corpus cavernosum penis.
frequency of the differentiation of corpus cavernosum penis, hyaline cartilage and
membrane bone of the p-segment, and fibrocartilage of the d-segment of os penis
in transplants is summarized in Table 1.
Development of genital tubercular mesenchyme cultivated in recombination with
heterologous epithelia
Genital tubercular mesenchyme of 16-5-day foetuses was recombined with a
fragment of heterologous epithelia and transplanted. We then examined whether
Induction of os penis
141
heterologous epithelia can induce fibrocartilage of the d-segment of os penis.
Dorsal epidermis of 16-5-day foetuses, urinary bladder epithelium of 16-5-day
foetuses, and urinary bladder epithelium of adult rats induced fibrocartilage of the
d-segment with a high frequency. The development of corpus cavernosum penis,
hyaline cartilage and membrane bone of the p-segment in these transplants was
similar to that of transplants of mesenchyme of 16-5-day foetuses cultured in the
absence of epithelium (Table 2).
DISCUSSION
Mesenchymal cells of various organs in vertebrate embryos need inductive
interaction with epithelia to differentiate into cartilages and bones when cultured
in situ, in vivo, or in vitro (for a review, see Hall, 1983). The inductive effects of
epithelium have been reported in the differentiation of cartilages in limb bud
(Saunders, 1948; Gumpel-Pinot, 1972), scleral cartilage (Newsome, 1972), and
membrane bone in the mandible (Tyler & Hall, 1977). In these studies, the
inductive effects of epithelium were demonstrated by in situ (Saunders, 1948),
in vivo (Newsome, 1972; Tyler & Hall, 1977), and in vitro organ-culture experiments (Newsome, 1976; Tyler & Hall, 1977; Gumpel-Pinot, 1980), suggesting that
the epithelium also exercises an inductive action during normal development. In
some of these experiments, heterologous epithelia are also active to induce
cartilages or bones (Gumpel-Pinot, 1972; Hall, 1981). It has been suggested that
the action of epithelium on progenitor cells of cartilage and bone is mediated by
non-diffusible epithelial cell products (Newsome, 1976; Gumpel-Pinot, 1980; Hall
& Van Exan, 1982; Hall, Van Exan & Brunt, 1983; Smith & Thorogood, 1983; Van
Exan & Hall, 1984). In spite of these studies, the role of epithelium in normal
development of cartilages and bones is not established yet, since in some culture
conditions epithelium inhibits chondrogenesis of mesenchymal cells (Solursh,
Singley & Reiter, 1981; Solursh et al 1984).
In the present study, we demonstrated that progenitor cells of the corpus
cavernosum penis and the os penis also need the presence of epithelium to acquire
potency of differentiation. Epithelium is necessary only in the stages before 17-5
days of gestation. At about 17-5-18-5 days of gestation, all the rudiments of the
corpus cavernosum penis, the p-segment of os penis, and the d-segment of os penis
became clearly recognizable as dense mesenchymal cell masses (Murakami &
Mizuno, 1984a). As described in Results, neither the cartilages and bone of the os
penis nor even the rudiments of these tissues were formed when genital tubercular
mesenchymes of the 14-5- to 16-5-day foetuses were cultured in the absence of
epithelium. Androgens are necessary for phenotypic differentiation of the
cartilages and bone of the os penis and the corpus cavernosum penis, while the
rudiments of these tissues can be formed without androgens (Murakami, in
preparation). These results strongly suggest that epithelium is necessary for the
formation of the rudiments, which are recognizable as dense mesenchymal cell
masses, that the rudiment formation is an indispensable step in the differentiation
142
R. MURAKAMI AND T. M I Z U N O
of the os penis and the corpus cavernosum penis, and that androgens cause the
phenotypic differentiation of the rudiment cells. The epithelium may also
stimulate the proliferation of the mesenchymal cells, since mesenchymes cultured
with epithelium became much larger than mesenchymes cultured without
epithelium. The heterologous epithelia, such as foetal dorsal epidermis and
urinary bladder epithelium of foetal and adult rats, could induce the d-segment of
os penis, but they could not promote differentiation of the membrane bone of
the p-segment as markedly as did homologous epithelium. The epithelialmesenchymal interaction in heterologous recombinates might be incomplete. In
any case, the inductive effect of epithelium on genital tubercular mesenchyme
seems to be a permissive one, because all heterologous epithelia examined could
induce differentiation of the d-segment of os penis in the mesenchyme.
In the development of chick limb bud, removal of AER at a certain stage causes
truncation of the limb at a particular level: the later the removal, the more distal
the truncation level (Saunders, 1948; Summerbell, 1974). From the results
of AER-removal experiments and carbon-marking experiments, Saunders concluded that future wing parts are laid down in proximal-distal order and that AER
is essential in this process. In the genital tubercular epithelium of rats, no
structural resemblance to AER was found, and it is unknown yet how the
progenitor cells of the corpus cavernosum penis and os penis are developed and
arranged spatially. However, removal of the genital tubercular epithelium at a
certain stage causes truncation of the skeletal tissues in the penis at a particular
level, as described in Results: the later the epithelial removal, the more distal the
truncation level. From the phenomenal similarity between the response of the
limb bud and that of the genital tubercle to epithelial removal, a common process
of development is supposed to exist in both the organs: the progenitor cells of the
mesodermal tissues acquire differentiation potency in proximal-distal sequence,
and epithelium is essential for progenitor cells to acquire this differentiation
potency.
The authors wish to express their deep gratitude to the late Dr Alfred Glucksmann in
Cambridge for his encouragement and continuous interest in our works.
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